Methods of synthesizing heteromultimeric polypeptides in yeast using a haploid mating strategy

ABSTRACT

Methods are provided for the synthesis and secretion of recombinant proteins preferably large mammalian proteins or hetero-multimeric proteins at high levels and for prolonged time in polyploid, preferably diploid yeast. These methods use various mating competent yeast, including Pichia. In a preferred embodiment, a first expression vector is transformed into a first haploid cell; and a second expression vector is transformed into a second haploid cell. The transformed haploid cells, each individually synthesizing a non-identical polypeptide, are identified and then genetically crossed or fused. The resulting diploid strains are utilized to produce and secrete fully assembled and biologically functional hetero-multimeric protein.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 10/577,074filed on May. 7, 2007, which US patent application is the US nationalphase of PCT/US/04/35302 which was published as PCT/WO/2005040395 filedon Oct. 22, 2004, which in turn claims benefit of priority toprovisional patent application U.S. Ser. No. 60/513,876 filed on Oct.22, 2003. All of these patent applications are expressly incorporated byreference in their entireties herein.

BACKGROUND OF THE INVENTION

Recombinant protein production is an essential activity for highthroughput screening, functional validation, structural biology, andproduction of pharmaceutical polypeptides. Escherichia coli a widelyused organism for the expression of heterologous proteins because iteasily grows to a high cell density on inexpensive substrates, and haswell-established genetic techniques and expression vectors. However,this is not always sufficient for the efficient production of activebiomolecules. In order to be biologically active, polypeptide chainshave to fold into the correct native three-dimensional structure,including the appropriate formation of disulfide bonds, and may furtherrequire correct association of multiple chains.

Although the active state of the protein may be thermodynamicallyfavored, the time-scale for folding can vary from milliseconds to days.Kinetic barriers are introduced, for example, by the need for alignmentof subunits and sub-domains. And particularly with eukaryotic proteins,covalent reactions must take place for the correctly folded protein toform. The latter types of reaction include disulfide bond formation,cis/trans isomerization of the polypeptide chain around proline peptidebonds, preprotein processing and the ligation of prosthetic groups.These kinetic limitations can result in the accumulation of partiallyfolded intermediates that contain exposed hydrophobic ‘sticky’ surfacesthat promote self-association and the formation of aggregates.

The expression of multimeric proteins such as hormones and enzymes inactive form in particular is difficult to achieve in some recombinantexpression systems. Many host cells do not possess the appropriateenzymes to process such proteins in biologically active form. Because ofthese difficulties such multimeric proteins usually must be expressed inmammalian cells. This unfortunately raises the costs associated withproducing the protein as well as resulting in issues of viralcontamination which are particularly problematic if the multimericprotein is to be used a therapeutic. One prevalent example of amultimeric protein typical produced in mammalian culture systems such asCHO cells is recombinant immunoglobulins or antibodies.

Antibodies are tetrameric proteins, which have many uses in clinicaldiagnosis and therapy. Each antibody tetramer is composed of twoidentical light chains and two identical heavy chains. Pure human orhumanized antibodies of a specific type are difficult or impossible topurify in sufficient amounts for many purposes from natural sources. Asa consequence, biotechnology and pharmaceutical companies have turned torecombinant DNA-based methods to prepare them on a large scale. Theproduction of functional antibodies requires not just the synthesis ofthe two polypeptides but also a number of post-translationalmodifications, including proteolytic processing of the N-terminalsecretion signal sequence; proper folding and assembly of thepolypeptides into tetramers; formation of disulfide bonds; and specificN-linked glycosylation. All of these events take place in the eukaryoticcell secretory pathway, an organelle complex unique to eukaryotic cells.

Recombinant synthesis of such complex proteins has had to rely on highereukaryotic tissue culture-based systems for biologically activematerial. However, as mentioned mammalian tissue culture basedproduction systems are significantly more expensive and complicated thanmicrobial fermentation methods. In addition, there continues to bequestions regarding therapeutic products produced using materialsderived from animal by-products.

Alternatives to mammalian expression systems for the expression ofrecombinant proteins are eukaryotic microbia such as yeast and insectcell expression systems. Insect cell expression systems use baculovirusvectors and often achieve high yields of secreted proteins. However,such cells are not always capable of processing complex mammalianpolypeptides in active form. Yeast find fairly well established usage inexpressing recombinant proteins. The most typically used yeast isSaccharomyces since it has been well characterized, many promoterssuitable for use therein are widely available as are sequences forfacilitating secretion such as the alpha and A factor secretory signalsequences. Another yeast which has been suggested to be capable ofproducing mammalian proteins in active form is Pichia, and particularlyPichia pastoris. As a eukaryote, Pichia pastoris has many of theadvantages of higher eukaryotic expression systems such as proteinprocessing, protein folding, and posttranslational modification, whilebeing as easy to manipulate as E. coli or Saccharomyces cerevisiae. Itis faster, easier, and less expensive to use than other eukaryoticexpression systems such as baculovirus or mammalian tissue culture, andgenerally gives higher expression levels. As a yeast, it shares theadvantages of molecular and genetic manipulations with Saccharomyces.These features make Pichia very useful as a protein expression system.In addition various other types of yeast have been disclosed to besuitable for expression of heterologous polypeptides.

Many of the techniques developed for Saccharomyces may be applied toPichia as well as to other types of yeast. These include transformationby complementation; gene disruption and gene replacement. In addition,the genetic nomenclature used for Saccharomyces been applied to Pichia.There is also cross-complementation between gene products in bothSaccharomyces and Pichia. Several wild-type genes from Saccharomycescomplement comparable mutant genes in Pichia.

Heterologous expression in Pichia pastoris as well as other types ofyeast can be either intracellular or secreted. Secretion requires thepresence of a signal sequence on the expressed protein to target it tothe secretory pathway. While several different secretion signalsequences have been used successfully, including the native secretionsignal present on some heterologous proteins, success has been variable.A potential advantage to secretion of heterologous proteins is thatPichia pastoris secretes very low levels of native proteins. That,combined with the very low amount of protein in the minimal Pichiagrowth medium, means that the secreted heterologous protein comprisesthe vast majority of the total protein in the medium and serves as thefirst step in purification of the protein.

Many species of yeast, including Pichia are mating competent. Thisenables two distinct haploid strains to mate naturally and generate adiploid species possessing two chromosomal copies Alternatively,polyploid yeast can be obtained by artificial methods, i.e., spheroplastfusion.

As noted yeast including Pichia have been used many years for theproduction of heterologous proteins. Although P. pastoris in particularhas been used successfully for the production of various heterologousproteins, e.g., hepatitis B surface antigen (Cregg et al. (1987)Bio/Technology 5:479), lysozyme and invertase (Digan et al. (1988) Dev.Indust. Micro. 29:59; Tschopp et al. (1987) Bio/Technology 5:1305),endeavors to produce other heterologous gene products in Pichia,especially by secretion, have given mixed results. At the present levelof understanding of the P. pastoris expression system, it isunpredictable whether a given gene can be expressed to an appreciablelevel in this yeast or whether Pichia will tolerate the presence of therecombinant gene product in its cells. Further, it is especiallydifficult to foresee if a particular protein will be secreted by P.pastoris, and if it is, at what efficiency.

Additionally, prior to the present invention the use of diploid yeast tosecrete heterologous polypeptides had not been reported. Rather, theearlier work using yeast to produce secreted heterologous polypeptideswas limited to haploidal yeast expression systems. In fact, earlierevidence suggested that diploid yeast such as Pichia would be incapableof stably expressing and secreting heterologous polypeptides in amountsrequired for such expression systems to be suitable for commercial use.

The present invention therefore provides improved methods andcompositions of matter that provide for the secretion of heterologouspolypeptides, preferably heteromultimers using polyploidal yeastcultures preferably produced from mating competent yeast, includingPichia and other genera.

OBJECTS OF THE INVENTION AND PREFERRED EMBODIMENTS:

It is an object of the invention to provide polyploid yeast cultureswhich stably express and secrete into the culture medium at least oneheterologous polypeptide in amounts of at least 10-25 mg/liter, morepreferably at least 25-100 mg/liter, still more preferably 100-250mg/liter, even more preferably 250-500 mg/liter, yet more preferably500-1000 mg/liter and most preferably in excess of 1 g/liter. Thesecultures will preferably stably express the polypeptide at such levelsfor at least several days to a week, more preferably at least a month,still more preferably at least 1-6 months, and most preferably up to ayear or longer

It is another object of the invention to provide a method for producinga polyploid yeast culture that stably expresses and secretes into theculture medium at lest 10-25 mg/liter of at least one heterologouspolypeptide comprising: (i) introducing a first expression vectorcontaining a heterologous DNA that encodes a first desired polypeptideoperably linked to a promoter and a signal sequence into a haploid cell;(ii) optionally introducing into a second haploid yeast cell a secondexpression vector that contains a heterologous DNA which encodes asecond heterologous polypeptide also operably linked to a promoter and asignal sequence (iii) producing by mating or spheroplast fusion apolyploid yeast from said first and/or second haploid yeast cells; (iv)selecting polyploid yeast cells that express and secrete said firstand/or second polypeptide; and (v) deriving polyploid yeast culturestherefrom that stably or for prolonged periods express in secreted format least 10-25 mg/liter of said first and/or second polypeptide. Inpreferred embodiments the amount of secreted polypeptide will range fromat least 25-100 mg/liter, more preferably 100-500 mg/liter, still morepreferably 500-1000 mg/liter or more.

It is another preferred embodiment of the invention to provide a methodfor producing a diploid yeast that stable expresses at least 10-25mg/liter of at least one desired heterologous polypeptide comprising:(i) introducing into a diploid yeast at least one desired heterologouspolypeptide; (ii) selecting diploid yeast cells which stable expresssaid at least one heterologous polypeptide; and (iii) generating adiploid culture therefrom that stably or for prolonged periods secretesat least 10-25 mg/liter of said at least one heterologous polypeptide.More preferably the yeast culture will express at least 25-50 mg/liter,still more preferably at least 50-250 mg/liter, even more preferably atleast 250-500 mg/liter and most preferably at least 500-1000 mg/liter ormore.

SUMMARY OF THE INVENTION

Methods are provided for the synthesis and secretion of recombinantpolypeptides in polyploid yeast cells, preferably diploid Pichia yeastor other mating competent yeast cells. Most typically such recombinantproteins will comprise mammalian polypeptides which are to be usedtherapeutically such as recombinant enzymes, hormones, growth factors,cytokines or lymphokines, cytotoxins or lymphotoxins, immunoglobulins,tumor antigens, receptors, and the like. More preferably such proteinswill comprise large mammalian proteins, i.e., at least proteins whichare at least several hundred amino acids or even in excess of 1000 aminoacids and will possess one or more cysteine residues and/or areglycosylated when expressed endogenously. Examples of such proteinsinclude lymphokines, enzymes, growth factors, lymphokines or cytokines,tumor antigens, viral antigens, immunotoxins and cytotoxins, and thelike. In an especially preferred embodiment the yeast expressed proteinwill be suitable for human therapy.

In a more preferred embodiment, the invention expresses secretedhomo-polymeric or hetero-multimeric proteins in diploid yeast,preferably mating competent yeast, more preferably Pichia.Hetero-multimeric proteins of interest comprise at least twonon-identical polypeptide chains, e.g. antibody heavy and light chains,MHC alpha and beta chains; heteropolymeric hormones, receptorpolypeptides, and the like. For example such heteropolymeric proteinsmay comprise heteropolymeric G protein coupled receptors.

In practicing this embodiment of the invention expression vectors arepreferably provided for the expression of each non-identical polypeptidechain. Alternatively, nucleic acid sequences encoding the non-identicalpolypeptide chains may be comprised on the same expression vector. Suchexpression vectors may be extrachromosomal or may be integrated into thediploid yeast cell's chromosomal DNA.

In the preferred embodiment a different expression vector is introducedinto two different haploid yeast cells. Alternatively, an expressionvector encoding one or more heterologous polypeptides, preferably thosewhich associate to produce a heteropolymeric polypeptide having adesired functionality may be introduced into a single haploid yeast cellwhich is then mated or fused with another yeast cell to produce adiploid or polyploidal yeast cell. Still alternatively a vector orvectors encoding providing for the secretion of the desired polypeptidechains may be introduced into a haploid yeast cell and this cell fusedor mated with another haploid cell to produce a diploid or tetraploidcell that expresses and secretes these polypeptides. Yet alternativelydiploid yeast may be transformed with one or more vectors containinggenes encoding and providing for the secretion of a desired heterologouspolypeptide.

In some embodiments of the invention, the haploid yeast cell isgenetically marked, where the haploid yeast cell is one of acomplementary pair. A first expression vector is transformed into onehaploid cell and a second expression vector is transformed into a secondhaploid cell. Where the haploid cells are to be mated this will bethrough direct genetic fusion, or a similar event is induced withspheroplast fusion.

The haploid yeast cells used for producing diploid or other polyploidalyeast cells expression may contain one or more genetic mutations whichresult in enhanced growth characteristics or enhanced secretory capacityor other improved phenotypic characteristic relative to yeast cellslacking the mutations. Yeast strains possessing such enhancecharacteristics are commercially available. Alternatively yeast cellsmay be mutagenized by known methods such as site specific mutagenesis orrandomly using e.g., UV irradiation or the use of chemical mutagens andthe resultant cultures screened to identify yeast strains possessingenhanced characteristics such as enhanced growth, stability, resistanceto pathogens, or secretory capabilities relative to non-mutant strains.

The expression levels of the non-identical polypeptides in the haploidor diploid yeast cells may be individually calibrated, and adjustedthrough appropriate selection, vector copy number, promoter strengthand/or induction and the like. In one embodiment of the invention, thepromoter in each expression vector or on the same vector for expressingthe different polypeptide subunits is different. Alternatively, if apolycistronic vector is used the positioning of the different genes onthe construct may be varied (flipped) in order to favor the expressionof one polypeptide relative to the other. In another embodiment of theinvention, the same promoter is provided for the expression of eachpolypeptide. Promoters may be constitutive or inducible. The types ofpromoters that are useful in the invention include promoters fromeukarotes such as yeast, insect, mammalian, viral, plant, fungal,amphibian, avian, and reptile sources. In general the promoters willcomprise yeast, mammalian, or viral promoters. Most typically thepromoters will be yeast promoters of highly expressed proteins.

In a preferred embodiment, the transformed haploid cells, eachindividually synthesizing a non-identical polypeptide, are identifiedand then genetically crossed or fused. The resulting diploid strains areutilized to produce and secrete fully assembled and biologicallyfunctional hetero-multimeric protein. The diploid methodology allowsoptimized subunit pairing to enhance full-length product generation andsecretion.

However, as mentioned, the invention contemplates the expression andsecretion of non-multimeric proteins in diploidal yeast cultures aswell, especially large mammalian proteins, i.e., at least severalhundred amino acids or even in excess of a thousand amino acids. Asnoted above, prior to the present invention experts in the field hadbelieved that diploid yeast would be unsuitable for achieving stableand/or prolonged expression and secretion of recombinant polypeptides,especially large heterologous polypeptides in amounts sufficient to becommercially viable. By contrast the present inventors have surprisinglydiscovered that diploidal yeast cultures express and secrete into theculture medium for prolonged periods, i.e., from at least several daysto at least a month or more, even for more than a year, amounts ofrecombinant polypeptide ranging from at least 10-25 mg/liter, 25-250mg/liter, 250-500 mg/liter, 500-1000 mg/liter and even amounts in excessof 1 gram/liter. These results are truly unexpected.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D. Generation of assembled full length recombinant antibody.Immunoblot detection methodology was used to characterize the parentalhaploid Pichia strains, each producing a subunit of the antibody andtarget diploid strain producing both subunits that form the fullyassembled antibody. The yeast strains shown in FIG. 1A show a staticculture of each of the representative strains, where the top portion isthe distinct haploids strains containing Heavy (H) and Light (L) chainsubunits respectively; the bottom the mated stable diploid producingboth subunits. FIG. 1B shows selective detection of the H chain, whichis found only in the parental H chain haploid, and mated diploidcontaining both H and L. FIG. 1C shows general detection of H & Lchains, which establishes that protein production is active in all threestrains. FIG. 1D shows selective detection in the diploid strain ofcorrectly assembled full antibody, confirming that only the diploidsystem is capable of generating fully assembled antibody.

FIG. 2. Full length antibody production in Pichia pastoris. Heterologousexpression of full-length antibody was conducted using a diploid Pichiapastoris strain. Exported antibody protein was isolated from conditionedmedia using Protein A affinity chromatography. An aliquot of the peakfraction is shown. The human IgG standard was derived from purifiedpooled human IgG.

FIG. 3. Assembled antibody was detected and characterized from mediasupernatants from subclones of diploid Pichia pastoris strains, whichwere engineered to produce full-length mouse/human chimeric antibody.Microtiter plates were coated with Anti-human Fc selective antibodies tocapture the antibody from the culture media. Correctly assembledantibody was detected through the use of a human selective (Fab′)2,which recognized the paired heavy CH1 and κ light chain constantregions. Serial dilutions of clarified media were applied to the plate.Development was through standard ELISA visualization methods. Thedetection is selective as shown by the lack of any detectable signal inthe mIgG standard.

FIG. 4. Pichia generated recombinant antibody stains CD3 containingJurkat T-cells as well as traditional mammalian-derived antibody. JurkatT-cells were immobilized on glass slides and staining was conductedusing the anti-CD3 antibody generated in yeast and mammalian cells.Detection was performed using a biotinylated-conjugated anti-rodentsecondary antibody, and developed with an HRP-streptavidin derivative.The imagines are representative field of slide treated with eachrecombinant antibody. Background is control for development andconducted in the absence of the primary anti-CD3 antibody.

DETAILED DESCRIPTION OF THE INVENTION

Recombinant polypeptides, preferably large mammalian polypeptides ormultimeric hetero-multimeric proteins are secreted from polyploidal,preferably diploid or tetraploid strains of mating competent yeast. Theinvention is directed to methods for producing recombinant polypeptidesin secreted form for prolonged periods using cultures comprisingpolyploid yeast i.e. at least several days to a week, more preferably atleast a month or several months, and even more preferably at least 6months to a year or longer. These polyploid yeast cultures will expressat least 10-25 mg/liter of the polypeptide, more preferably at least50-250 mg/liter, still more preferably at least 500-1000 mg/liter, andmost preferably a gram per liter or more of the recombinant polypeptide.The methods are especially useful for producing large mammalian proteinsand multimeric polypeptides.

In one embodiment of the invention a pair of genetically marked yeasthaploid cells are transformed with expression vectors comprisingsubunits of a desired heteromultimeric protein. One haploid cellcomprises a first expression vector, and a second haploid cell comprisesa second expression vector. In another embodiment diploid yeast cellswill be transformed with one or more expression vectors that provide forthe expression and secretion of one or more recombinant polypeptides. Instill another embodiment a single haploid cell may be transformed withone or more vectors and used to produce a polyploidal yeast by fusion ormating strategies. In yet another embodiment a diploid yeast culture maybe transformed with one or more vectors providing for the expression andsecretion of a desired polypeptide or polypeptides. These vectors maycomprise plasmids that are maintained extrachromosomally or may comprisevectors e.g., linearized plasmids that integrate into the yeast cell'sgenome randomly or by homologous recombination. Optionally, additionalexpression vectors may be introduced into the haploid or diploid cells;or the first or second expression vectors may comprise additional codingsequences; for the synthesis of heterotrimers; heterotetramers; etc. Theexpression levels of the non-identical polypeptides may be individuallycalibrated, and adjusted through appropriate selection, vector copynumber, promoter strength and/or induction and the like. The transformedhaploid cells are genetically crossed or fused. The resulting diploid ortetraploid strains are utilized to produce and secrete fully assembledand biologically functional proteins preferably hetero-multimericproteins.

The use of diploid or tetraploid cells for protein production providesfor unexpected benefits. The cells can be grown for production purposes,i.e. scaled up, and for extended periods of time, in conditions that canbe deleterious to the growth of haploid cells, which conditions mayinclude high cell density; growth in minimal media; growth at lowtemperatures; stable growth in the absence of selective pressure; andwhich may provide for maintenance of heterologous gene sequenceintegrity and maintenance of high level expression over time. Indeed theinventors have achieved expression yields in excess of about 1 g/literand these yields may be enhanced by further optimization. Withoutwishing to be bound thereby, the inventors theorize that these benefitsmay arise, at least in part, from the creation of diploid strains fromtwo distinct parental haploid strains. Such haploid strains can comprisenumerous minor autotrophic mutations, which mutations are complementedin the diploid or tetraploid, enabling growth under highly selectiveconditions.

DEFINITIONS

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,and reagents described, as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention which will be limited only by the appended claims.

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the protein” includes reference to one or more proteinsand equivalents thereof known to those skilled in the art, and so forth.All technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs unless clearly indicated otherwise.

Mating competent yeast species. In the present invention this isintended to broadly encompass any diploid yeast which can be stablymaintained in culture. Such species of yeast exist in a haploid and adiploid form. The diploid cells may, under appropriate conditions,proliferate for indefinite number of generations in the diploid form.Diploid cells can also sporulate to form haploid cells. In addition,sequential mating can result in tetraploid strains through furthermating of the auxotrophic diploids. In the present invention the diploidor polyploidal yeast cells are preferably produced by mating orspheroplast fusion.

In one embodiment of the invention, the mating competent yeast is amember of the Saccharomycetaceae family, which includes the generaArxiozyma; Ascoboryozyma; Citeromyces; Debaryomyces; Dekkera;Eremothecium; Issatchenkia; Kazachstania; Kluyveromyces; Kodamae;Lodderomyces; Pachysolen; Pichia; Saccharomyces; Satrunispora;Tetrapisispora; Torulaspora; Williopsis, and Zygosaccharomyces. Othertypes of yeast potentially useful in the invention include Yarrowia,Rhodosporidium, Candida, Hansenula, Filobasium, Filobasidellla,Sporidiobolus, Bullera, Leucosporidium and Filobasidella.

The genus Pichia is of particular interest. Pichia comprises a number ofspecies, including the species Pichia pastoris, Pichia methanolica, andHansenula polymorpha (Pichia angusta). Most preferred is the speciesPichia pastoris.

Haploid Yeast Cell. A cell having a single copy of each gene of itsnormal genomic (chromosomal) complement.

Polyploid Yeast Cell A cell having more than one copy of its normalgenomic (chromosomal) complement.

Diploid Yeast Cell. A cell having two copies (alleles) of every gene ofits normal genomic complement, typically formed by the process of fusion(mating) of two haploid cells.

Tetraploid Yeast Cell. A cell having four copies (alleles) of every geneof its normal genomic complement, typically formed by the process offusion (mating) of two haploid cells. Tetraploids may carry two, three,or four different cassettes. Such tetraploids might be obtained in S.cerevisiae by selective mating homozygotic heterothallic a/a andalpha/alpha diploids and in Pichia by sequential mating of haploids toobtain auxotrophic diploids. For example, a [met his] haploid can bemated with [ade his] haploid to obtain diploid [his]; and a [met arg]haploid can be mated with [ade arg] haploid to obtain diploid [arg];then the diploid [his]×diploid [arg] to obtain a tetraploid prototroph.It will be understood by those of skill in the art that reference to thebenefits and uses of diploid cells may also apply to tetraploid cells.

Yeast Mating: The process by which two haploid yeast cells naturallyfuse to form one diploid yeast cell.

Meiosis: The process by which a diploid yeast cell undergoes reductivedivision to form four haploid spore products. Each spore may thengerminate and form a haploid vegetatively growing cell line.

Selectable Marker: A selectable marker is a gene or gene fragment thatconfers a growth phenotype (physical growth characteristic) on a cellreceiving that gene as, for example through a transformation event. Theselectable marker allows that cell to survive and grow in a selectivegrowth medium under conditions in which cells that do not receive thatselectable marker gene cannot grow. Selectable marker genes generallyfall into several types, including positive selectable marker genes suchas a gene that confers on a cell resistance to an antibiotic or otherdrug, temperature when two ts mutants are crossed or a ts mutant istransformed; negative selectable marker genes such as a biosyntheticgene that confers on a cell the ability to grow in a medium without aspecific nutrient needed by all cells that do not have that biosyntheticgene, or a mutagenized biosynthetic gene that confers on a cellinability to grow by cells that do not have the wild type gene; and thelike. Suitable markers include but are not limited to: ZEO; G418; HIS 5;LYS3; MET1; MET3a; ADE1; ADE3; URA3; and the like.

Expression Vector: These DNA species contain elements that facilitatemanipulation for the expression of a foreign protein within the targethost cell. Conveniently, manipulation of sequences and production of DNAfor transformation is first performed in a bacterial host, e.g. E. coli,and usually vectors will include sequences to facilitate suchmanipulations, including a bacterial origin of replication andappropriate bacterial selection marker. Selectable markers encodeproteins necessary for the survival or growth of transformed host cellsgrown in a selective culture medium. Host cells not transformed with thevector containing the selection gene will not survive in the culturemedium. Typical selection genes encode proteins that (a) conferresistance to antibiotics or other toxins, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media.

Expression vectors for use in the methods of the invention will furtherinclude yeast specific sequences, including a selectable auxotrophic ordrug marker for identifying transformed yeast strains. A drug marker mayfurther be used to amplify copy number of the vector in a yeast hostcell.

The polypeptide coding sequence of interest is operably linked totranscriptional and translational regulatory sequences that provide forexpression of the polypeptide in yeast cells. These vector componentsmay include, but are not limited to, one or more of the following: anenhancer element, a promoter, and a transcription termination sequence.Sequences for the secretion of the polypeptide may also be included,e.g. a signal sequence, and the like. A yeast origin of replication isoptional, as expression vectors are often integrated into the yeastgenome.

In one embodiment of the invention, the polypeptide of interest isoperably linked, or fused, to sequences providing for optimizedsecretion of the polypeptide from yeast diploid cells.

Nucleic acids are “operably linked” when placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for asignal sequence is operably linked to DNA for a polypeptide if it isexpressed as a preprotein that participates in the secretion of thepolypeptide; a promoter or enhancer is operably linked to a codingsequence if it affects the transcription of the sequence. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inreading phase. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites oralternatively via a PCR/recombination method familiar to those skilledin the art (Gateway® Technology; Invitrogen, Carlsbad Calif.). If suchsites do not exist, the synthetic oligonucleotide adapters or linkersare used in accordance with conventional practice.

Promoters are untranslated sequences located upstream (5′) to the startcodon of a structural gene (generally within about 100 to 1000 bp) thatcontrol the transcription and translation of particular nucleic acidsequence to which they are operably linked. Such promoters fall intoseveral classes: inducible, constitutive, and repressible promoters thatincrease levels of transcription in response to absence of a repressor.Inducible promoters may initiate increased levels of transcription fromDNA under their control in response to some change in cultureconditions, e.g., the presence or absence of a nutrient or a change intemperature.

The yeast promoter fragment may also serve as the site for homologousrecombination and integration of the expression vector into the samesite in the yeast genome; alternatively a selectable marker is used asthe site for homologous recombination. Pichia transformation isdescribed in Cregg et al. (1985) Mol. Cell. Biol. 5:3376-3385.

Examples of suitable promoters from Pichia include the AOX1 and promoter(Cregg et al. (1989) Mol. Cell. Biol. 9:1316-1323); ICL1 promoter(Menendez et al. (2003) Yeast 20(13):1097-108);glyceraldehyde-3-phosphate dehydrogenase promoter (GAP) (Waterham et al.(1997) Gene 186(1):37-44); and FLD1 promoter (Shen et al. (1998) Gene216(1):93-102). The GAP promoter is a strong constitutive promoter andthe AOX and FLD1 promoters are inducible.

Other yeast promoters include GAPDH, ADH/GAP, alcohol dehydrogenase II,GAL4, PHO3, PHO5, Pyk, and chimeric promoters derived therefrom.Additionally, non-yeast promoters may be used in the invention such asmammalian, insect, plant, reptile , amphibian, viral, and avianpromoters. Most typically the promoter will comprise a mammalianpromoter (potentially endogenous to the expressed genes) or willcomprise a yeast or viral promoter that provides for efficienttranscription in yeast systems.

The polypeptides of interest may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, e.g. a signal sequence or other polypeptide having aspecific cleavage site at the N-terminus of the mature protein orpolypeptide. In general, the signal sequence may be a component of thevector, or it may be a part of the polypeptide coding sequence that isinserted into the vector. The heterologous signal sequence selectedpreferably is one that is recognized and processed through one of thestandard pathways available within the host cell. The S. cerevisiaealpha factor pre-pro signal has proven effective in the secretion of avariety of recombinant proteins from P. pastoris. Other yeast signalsequences include the mating factor A signal sequence, the invertasesignal sequence, and signal sequences derived from other secreted yeastpolypeptides. Additionally, these signal peptide sequences may beengineered to provide for enhanced secretion in diploid yeast expressionsystems. Other secretion signals of interest also include mammaliansignal sequences, which may be heterologous to the protein beingsecreted, or may be a native sequence for the protein being secreted.Signal sequences include pre-peptide sequences, and in some instancesmay include propeptide sequences. Many such signal sequences are knownin the art, including the signal sequences found on immunoglobulinchains, e.g. K28 preprotoxin sequence, PHA-E, FACE, human MCP-1, humanserum albumin signal sequences, human Ig heavy chain, human Ig lightchain, and the like. For example, see Hashimoto et. al. Protein Eng11(2) 75 (1998); and Kobayashi et. al. Therapeutic Apheresis 2(4) 257(1998).

Transcription may be increased by inserting a transcriptional activatorsequence into the vector. These activators are cis-acting elements ofDNA, usually about from 10 to 300 bp, which act on a promoter toincrease its transcription. Transcriptional enhancers are relativelyorientation and position independent, having been found 5′ and 3′ to thetranscription unit, within an intron, as well as within the codingsequence itself. The enhancer may be spliced into the expression vectorat a position 5′ or 3′ to the coding sequence, but is preferably locatedat a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells may also containsequences necessary for the termination of transcription and forstabilizing the mRNA. Such sequences are commonly available from 3′ tothe translation termination codon, in untranslated regions of eukaryoticor viral DNAs or cDNAs. These regions contain nucleotide segmentstranscribed as polyadenylated fragments in the untranslated portion ofthe mRNA.

Construction of suitable vectors containing one or more of theabove-listed components employs standard ligation techniques orPCR/recombination methods. Isolated plasmids or DNA fragments arecleaved, tailored, and relegated in the form desired to generate theplasmids required or via recombination methods. For analysis to confirmcorrect sequences in plasmids constructed, the ligation mixtures areused to transform host cells, and successful transformants selected byantibiotic resistance (e.g. ampicillin or Zeocin ) where appropriate.Plasmids from the transformants are prepared, analyzed by restrictionendonuclease digestion and/or sequenced.

As an alternative to restriction and ligation of fragments,recombination methods based on att sites and recombination enzymes maybe used to insert DNA sequences into a vector. Such methods aredescribed, for example, by Landy (1989) Ann. Rev. Biochem. 58.913-949;and are known to those of skill in the art. Such methods utilizeintermolecular DNA recombination that is mediated by a mixture of lambdaand E.coli-encoded recombination proteins. Recombination occurs betweenspecific attachment (att) sites on the interacting DNA molecules. For adescription of att sites see Weisberg and Landy (1983) Site-SpecificRecombination in Phage Lambda, in Lambda II, Weisberg, ed.(Cold SpringHarbor, N.Y.: Cold Spring Harbor Press), pp. 211-250. The DNA segmentsflanking the recombination sites are switched, such that afterrecombination, the att sites are hybrid sequences comprised of sequencesdonated by each parental vector. The recombination can occur betweenDNAs of any topology.

Att sites may be introduced into a sequence of interest by ligating thesequence of interest into an appropriate vector; generating a PCRproduct containing attB sites through the use of specific primers;generating a cDNA library cloned into an appropriate vector containingatt sites; and the like.

Folding, as used herein, refers to the three-dimensional structure ofpolypeptides and proteins, where interactions between amino acidresidues act to stabilize the structure. While non-covalent interactionsare important in determining structure, usually the proteins of interestwill have intra- and/or intermolecular covalent disulfide bonds formedby two cysteine residues. For naturally occurring proteins andpolypeptides or derivatives and variants thereof, the proper folding istypically the arrangement that results in optimal biological activity,and can conveniently be monitored by assays for activity, e.g. ligandbinding, enzymatic activity, etc.

In some instances, for example where the desired product is of syntheticorigin, assays based on biological activity will be less meaningful. Theproper folding of such molecules may be determined on the basis ofphysical properties, energetic considerations, modeling studies, and thelike.

The expression host may be further modified by the introduction ofsequences encoding one or more enzymes that enhance folding anddisulfide bond formation, i.e. foldases, chaperoning, etc. Suchsequences may be constitutively or inducibly expressed in the yeast hostcell, using vectors, markers, etc. as known in the art. Preferably thesequences, including transcriptional regulatory elements sufficient forthe desired pattern of expression, are stably integrated in the yeastgenome through a targeted methodology.

For example, the eukaryotic PDI is not only an efficient catalyst ofprotein cysteine oxidation and disulfide bond isomerization, but alsoexhibits chaperone activity. Co-expression of PDI can facilitate theproduction of active proteins having multiple disulfide bonds. Also ofinterest is the expression of BIP (immunoglobulin heavy chain bindingprotein); cyclophilin; and the like. In one embodiment of the invention,each of the haploid parental strains expresses a distinct foldingenzyme, e.g. one strain may express BIP, and the other strain mayexpress PDI.

The terms “desired protein” or “target protein” are used interchangeablyand refer generally to any secreted protein . Preferably this proteinwill comprise a large mamalian protein, typically at least 100 aminoacids, more typically 100-500 amino acids or over a thousand amino acidslong. In the exemplary embodiment, the desired protein will comprise aheteropolymeric protein comprised of 2 or more non-identical polypeptidechains, where such chains are independently synthesized, i.e. notresulting from post-translational cleavage of a single polypeptidechain. The polypeptides are heterologous, i.e., foreign, to the yeast.Preferably, mammalian polypeptides, i.e. polypeptides encoded in amammalian genome are used. Examples of such proteins include hormones,growth factors, cytokines, lymphokines, enzymes, receptors, cytotoxins,lymphotoxin, immunotoxins, tumor polypeptides, viral polypeptides,antibodies et al. These proteins or the corresponding gene may beengineered to facilitate expression in diploid yeast, e.g., by removalof glycosylation sites, removal of one or more cysteine residues, use ofyeast preferred codons, elimination of cleavage sites, and the like.

In a preferred embodiment, the protein is an antibody or a bindingportion thereof. The term “antibody” is intended to include anypolypeptide chain-containing molecular structure with a specific shapethat fits to and recognizes an epitope, where one or more non-covalentbinding interactions stabilize the complex between the molecularstructure and the epitope. The archetypal antibody molecule is theimmunoglobulin, and all types of immunoglobulins, IgG, IgM, IgA, IgE,IgD, etc., from all sources, e.g. human, rodent, rabbit, cow, sheep,pig, dog, other mammals, chicken, other avians, etc., are considered tobe “antibodies.” A preferred source for producing antibodies useful asstarting material according to the invention is rabbits. Numerousantibody coding sequences have been described; and others may be raisedby methods well-known in the art. Examples thereof include chimericantibodies, human antibodies and other non-human mammalian antibodies,humanized antibodies, single chain antibodies (scFvs), camelbodies,SNIPS, and antibody fragments such as Fabs, Fab′, Fab2 and the like.

For example, antibodies or antigen binding fragments may be produced bygenetic engineering. In this technique, as with other methods,antibody-producing cells are sensitized to the desired antigen orimmunogen. The messenger RNA isolated from antibody producing cells isused as a template to make cDNA using PCR amplification. A library ofvectors, each containing one heavy chain gene and one light chain generetaining the initial antigen specificity, is produced by insertion ofappropriate sections of the amplified immunoglobulin cDNA into theexpression vectors. A combinatorial library is constructed by combiningthe heavy chain gene library with the light chain gene library. Thisresults in a library of clones which co-express a heavy and light chain(resembling the Fab fragment or antigen binding fragment of an antibodymolecule). The vectors that carry these genes are co-transfected into ahost cell. When antibody gene synthesis is induced in the transfectedhost, the heavy and light chain proteins self-assemble to produce activeantibodies that can be detected by screening with the antigen orimmunogen.

Antibody coding sequences of interest include those encoded by nativesequences, as well as nucleic acids that, by virtue of the degeneracy ofthe genetic code, are not identical in sequence to the disclosed nucleicacids, and variants thereof. Variant polypeptides can include amino acid(aa) substitutions, additions or deletions. The amino acid substitutionscan be conservative amino acid substitutions or substitutions toeliminate non-essential amino acids, such as to alter a glycosylationsite, or to minimize misfolding by substitution or deletion of one ormore cysteine residues that are not necessary for function. Variants canbe designed so as to retain or have enhanced biological activity of aparticular region of the protein (e.g., a functional domain, catalyticamino acid residues, em. Variants also include fragments of thepolypeptides disclosed herein, particularly biologically activefragments and/or fragments corresponding to functional domains.Techniques for in vitro mutagenesis of cloned genes are known. Alsoincluded in the subject invention are polypeptides that have beenmodified using ordinary molecular biological techniques so as to improvetheir resistance to proteolytic degradation or to optimize solubilityproperties or to render them more suitable as a therapeutic agent.

Chimeric antibodies may be made by recombinant means by combining thevariable light and heavy chain regions (VK and VH), obtained fromantibody producing cells of one species with the constant light andheavy chain regions from another. Typically chimeric antibodies utilizerodent or rabbit variable regions and human constant regions, in orderto produce an antibody with predominantly human domains. The productionof such chimeric antibodies is well known in the art, and may beachieved by standard means (as described, e.g., in U.S. Pat. No.5,624,659, incorporated fully herein by reference).

Humanized antibodies are engineered to contain even more human-likeimmunoglobulin domains, and incorporate only thecomplementarity-determining regions of the animal-derived antibody. Thisis accomplished by carefully examining the sequence of thehyper-variable loops of the variable regions of the monoclonal antibody,and fitting them to the structure of the human antibody chains. Althoughfacially complex, the process is straightforward in practice. See, e.g.,U.S. Pat. No. 6,187,287, incorporated fully herein by reference.

In addition to entire immunoglobulins (or their recombinantcounterparts), immunoglobulin fragments comprising the epitope bindingsite (e.g., Fab′, F(ab′)₂, or other fragments) may be synthesized.“Fragment,” or minimal immunoglobulins may be designed utilizingrecombinant immunoglobulin techniques. For instance “Fv” immunoglobulinsfor use in the present invention may be produced by synthesizing avariable light chain region and a variable heavy chain region.Combinations of antibodies are also of interest, e.g. diabodies, whichcomprise two distinct Fv specificities.

Immunoglobulins may be modified post-translationally, e.g. to addchemical linkers, detectable moieties, such as fluorescent dyes,enzymes, substrates, chemiluminescent moieties and the like, or specificbinding moieties, such as streptavidin, avidin, or biotin, and the likemay be utilized in the methods and compositions of the presentinvention.

The term “polyploid yeast that stably expresses or expresses a desiredsecreted heterologous polypeptide for prolonged time” refers to a yeastculture that secretes said polypeptide for at least several days to aweek, more preferably at least a month, still more preferably at least1-6 months, and even more preferably for more than a year at thresholdexpression levels, typically at least 10-25 mg/liter and preferablysubstantially greater.

The term “polyploidal yeast culture that secretes desired amounts ofrecombinant polypeptide” refers to cultures that stably or for prolongedperiods secrete at least 10-25 mg/liter of heterologous polypeptide,more preferably at least 50-500 mg/liter, and most preferably 500-1000mg/liter or more.

METHODS OF POLYPEPTIDE SYNTHESIS

Transformed mating competent haploid yeast cells provide a geneticmethod that enables subunit pairing of a desired protein. Haploid yeaststrains are transformed with each of two expression vectors, a firstvector to direct the synthesis of one polypeptide chain and a secondvector to direct the synthesis of a second, non-identical polypeptidechain. The two haploid strains are mated to provide a diploid host whereoptimized target protein production can be obtained.

Optionally, additional non-identical coding sequence(s) are provided.Such sequences may be present on additional expression vectors or in thefirst or the second expression vectors. As is known in the art, multiplecoding sequences may be independently expressed from individualpromoters; or may be coordinately expressed through the inclusion of an“internal ribosome entry site” or “IRES”, which is an element thatpromotes direct internal ribosome entry to the initiation codon, such asATG, of a cistron (a protein encoding region), thereby leading to thecap-independent translation of the gene. IRES elements functional inyeast are described by Thompson et al. (2001) P.N.A.S. 98:12866-12868.

In one embodiment of the invention, antibody sequences are produced incombination with a secretary J chain, which provides for enhancedstability of IgA (see U.S. Pat. Nos. 5,959,177; and 5,202,422).

In a preferred embodiment the two haploid yeast strains are eachauxotrophic, and require supplementation of media for growth of thehaploid cells. The pair of auxotrophs are complementary, such that thediploid product will grow in the absence of the supplements required forthe haploid cells. Many such genetic markers are known in yeast,including requirements for amino acids (e.g. met, lys, his, arg, etc.),nucleosides (e.g. ura3, ade1, etc.); and the like. Amino acid markersmay be preferred for the methods of the invention. Alternatively diploidcells which contain the desired vectors can be selected by other means,e.g., by use of other selectable markers, such as green fluorescentprotein, various dominant selectable markers, and the like.

The two transformed haploid cells may be genetically crossed and diploidstrains arising from this mating event selected by their hybridnutritional requirements. Alternatively, populations of the twotransformed haploid strains are spheroplasted and fused, and diploidprogeny regenerated and selected. By either method, diploid strains canbe identified and selectively grown because, unlike their haploidparents, they do not have the same nutritional requirements. Forexample, the diploid cells may be grown in minimal medium. The diploidsynthesis strategy has certain advantages. Diploid strains have thepotential to produce enhanced levels of heterologous protein throughbroader complementation to underlying mutations, which may impact theproduction and/or secretion of recombinant protein.

As noted above, in some embodiments a haploid yeast may be transformedwith a single or multiple vectors and mated or fused with anon-transformed cell to produce a diploid cell containing the vector orvectors. In other embodiments, a diploid yeast cell may be transformedwith one or more vectors that provide for the expression and secretionof a desired heterologous polypeptide by the diploid yeast cell.

In one embodiment of the invention, two haploid strains are transformedwith a library of polypeptides, e.g. a library of antibody heavy orlight chains. Transformed haploid cells that synthesize the polypeptidesare mated with the complementary haploid cells. The resulting diploidcells are screened for functional protein. The diploid cells provide ameans of rapidly, conveniently and inexpensively bringing together alarge number of combinations of polypeptides for functional testing.This technology is especially applicable for the generation ofheterodimeric protein products, where optimized subunit synthesis levelsare critical for functional protein expression and secretion.

In another embodiment of the invention, the expression level ratio ofthe two subunits is regulated in order to maximize product generation.Heterodimer subunit protein levels have been shown previously to impactthe final product generation (Simmons L C, J Immunol Methods. May 1,2002; 263(1-2):133-47). Regulation can be achieved prior to the matingstep by selection for a marker present on the expression vector. Bystably increasing the copy number of the vector, the expression levelcan be increased. In some cases, it may be desirable to increase thelevel of one chain relative to the other, so as to reach a balancedproportion between the subunits of the polypeptide. Antibioticresistance markers are useful for this purpose, e.g. Zeocin resistancemarker, G418 resistance, etc. and provide a means of enrichment forstrains that contain multiple integrated copies of an expression vectorin a strain by selecting for transformants that are resistant to higherlevels of Zeocin or G418. The proper ratio, e.g. 1:1; 1:2; etc, of thesubunit genes may be important for efficient protein production. Evenwhen the same promoter is used to transcribe both subunits, many otherfactors contribute to the final level of protein expressed andtherefore, it can be useful to increase the number of copies of oneencoded gene relative to the other. Alternatively, diploid strains thatproduce higher levels of a polypeptide, relative to single copy vectorstrains, are created by mating two haploid strains, both of which havemultiple copies of the expression vectors.

Host cells are transformed with the above-described expression vectors,mated to form diploid strains, and cultured in conventional nutrientmedia modified as appropriate for inducing promoters, selectingtransformants or amplifying the genes encoding the desired sequences. Anumber of minimal media suitable for the growth of yeast are known inthe art. Any of these media may be supplemented as necessary with salts(such as sodium chloride, calcium, magnesium, and phosphate), buffers(such as HEPES), nucleosides (such as adenosine and thymidine),antibiotics, trace elements, and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

Secreted proteins are recovered from the culture medium. A proteaseinhibitor, such as phenyl methyl sulfonyl fluoride (PMSF) may be usefulto inhibit proteolytic degradation during purification, and antibioticsmay be included to prevent the growth of adventitious contaminants. Thecomposition may be concentrated, filtered, dialyzed, etc., using methodsknown in the art.

The diploid cells of the invention are grown for production purposes.Such production purposes desirably include growth in minimal media,which media lacks pre-formed amino acids and other complex biomolecules,e.g. media comprising ammonia as a nitrogen source, and glucose as anenergy and carbon source, and salts as a source of phosphate, calciumand the like. Preferably such production media lacks selective agentssuch as antibiotics, amino acids, purines, pyrimidines, etc. The diploidcells can be grown to high cell density, for example at least about 50g/L; more usually at least about 100 g/L; and may be at least about 300,about 400, about 500 g/L or more.

In one embodiment of the invention, the growth of the subject cells forproduction purposes is performed at low temperatures, which temperaturesmay be lowered during log phase, during stationary phase, or both. Theterm “low temperature” refers to temperatures of at least about 15° C.,more usually at least about 17° C., and may be about 20° C., and isusually not more than about 25° C., more usually not more than about 22°C. Growth temperature can impact the production of full-length secretedproteins in production cultures, and decreasing the culture growthtemperature can strongly enhances the intact product yield. Thedecreased temperature appears to assist intracellular traffickingthrough the folding and post-translational processing pathways used bythe host to generate the target product, along with reduction ofcellular protease degradation.

The methods of the invention provide for expression of secreted, activeprotein, preferably a mammalian protein. In the preferred particularlysecreted, active antibodies, where “active antibodies”, as used herein,refers to a correctly folded multimer of at least two properly pairedchains, which accurately binds to its cognate antigen. Expression levelsof active protein are usually at least about 10-50 mg/liter culture,more usually at least about 100 mg/liter, preferably at least about 500mg/liter, and may be 1000 mg/liter or more.

The methods of the invention can provide for increased stability of thehost and heterologous coding sequences during production. The stabilityis evidenced, for example, by maintenance of high levels of expressionof time, where the starting level of expression is decreased by not morethan about 20%, usually not more than 10%, and may be decreased by notmore than about 5% over about 20 doublings, 50 doublings, 100 doublings,or more.

The strain stability also provides for maintenance of heterologous genesequence integrity over time, where the sequence of the active codingsequence and requisite transcriptional regulatory elements aremaintained in at least about 99% of the diploid cells, usually in atleast about 99.9% of the diploid cells, and preferably in at least about99.99% of the diploid cells over about 20 doublings, 50 doublings, 100doublings, or more. Preferably, substantially all of the diploid cellsmaintain the sequence of the active coding sequence and requisitetranscriptional regulatory elements.

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,constructs, and reagents described, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention, which will be limited onlyby the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing, for example, the celllines, constructs, and methodologies that are described in thepublications, which might be used in connection with the presentlydescribed invention. The publications discussed above and throughout thetext are provided solely for their disclosure prior to the filing dateof the present application. Nothing herein is to be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention. Efforts have been made toensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric.

EXPERIMENTAL Example 1

To demonstrate the efficacy of the diploid antibody production methodthe following reagents were prepared.

Antibody genes: Genes were cloned and constructed that directed thesynthesis of three forms of a chimeric humanized mouse monoclonalantibody OKT3. The sources of the variable regions for use in theseconstructs can be found in Genbank. Accession number A22261; mouse OKT3heavy chain (International Patent Application WO 9109967-A 3 11 Jul.1991). Accession number A22259; mouse OKT3 light chain (InternationalPatent Application WO 9109967-A 3 11Jul. 1991).

All three forms utilized the identical V_(κ)C_(κ)light chain gene. Forthe three heavy chain genes, all encoded the identical mouse variableregion (V_(h)) but differed from each other in the amino acid sequenceof the human heavy chain constant regions. The first construct directedthe synthesis of a full-length wild-type heavy chain (C_(γ1)) with itssingle normal N-linked glycosylation site present (full-lengthglycosylated heavy chain). The second gene directed the synthesis of anon-glycosylated heavy chain created by mutating a nucleotide in thesequence so that a threonine at postion 301 was changed to an alanine inthe glycosylation site recognition sequence (Asn-X-Thr/Ser) (full-lengthnon-glycosylated heavy chain). The third gene construct directed thesynthesis of a heavy chain in which most of the constant region wasdeleted after the hinge region (Fab heavy chain).

Expression vector: The vector contains the following functionalcomponents: 1) a mutant ColE1 origin of replication, which facilitatesthe replication of the plasmid vector in cells of the bacteriumEscherichia coli, 2) a bacterial Sh ble gene, which confers resistanceto the antibiotic Zeocin and serves as the selectable marker fortransformations of both E. coli and P. pastoris, 3) an expressioncassette composed of the glyceraldehyde dehydrogenase gene (GAP gene)promoter, fused to sequences encoding the Saccharomyces cerevisiae alphamating factor pre pro secretion leader sequence, followed by sequencesencoding a P. pastoris transcriptional termination signal from the P.pastoris alcohol oxidase I gene (AOX1). The Zeocin resistance markergene provides a means of enrichment for strains that contain multipleintegrated copies of an expression vector in a strain by selecting fortransformants that are resistant to higher levels of Zeocin.

P. pastoris strains: The auxotrophic strains used for this example arethe P. pastoris ade1 and ura3 strains, which require supplementationwith adenine and uracil, respectively, for growth. Strains met1 and tys3have also been used. Although any two complementing sets of auxotrophicstrains could be used for the construction and maintenance of diploidstrains, these two strains are especially suited for this method for tworeasons. First, they grow more slowly than diploid strains that are theresult of their mating or fusion. Thus, if a small number of haploidade1 or ura3 cells remain present in a culture or arise through meiosisor other mechanism, the diploid strain should outgrow them in culture.

The second is that it is easy to monitor the sexual state of thesestrains since colonies of the diploid product of their mating are anormal white or cream color, whereas cells of any strains that arehaploid ade1 mutants in a culture form a colony with distinct pink incolor. In addition, any strains that are haploid ure3 mutants areresistant to the drug 5-fluoro-orotic acid (FOA) and can be sensitivelyidentified by plating samples of a culture on minimal medium+uracilplates with FOA. On these plates, only uracil-requiring ura3 mutant(presumably haploid) strains can grow and form colonies. Thus, withhaploid parent strains marked with ade1 and ura3, one can readilymonitor the sexual state of the resulting antibody-producing diploidstrains (haploid versus diploid).

Methods

Construction of pGAPZ-alpha expression vectors for transcription oflight and heavy chain antibody genes. For cloning of both the light andheavy chain variable regions, cells of a mouse OKT3 CD3 hybridoma cellline were grown and total RNA extracted. Two RT-PCR reactions were thenperformed, one specific to light and one specific to heavy chainvariable region encoding sequences of the OKT3 antibody genes. Theprimers employed to amplify out the heavy and light chain variableregion were (SEQ ID NO:1)5′-CCGCTCGAGAAAAGAGAGGCTGAAGCTCAGGTCCAGCTGCAGCAGTC-3′ and (SEQ ID NO:3)5′-CCGCTCGAGAAAAGAGAGGCTGAAGCTCAAATTGTTCTCACCCAGTCTCC-3′ along with (SEQID NO:2) 5′-TGGGCCCTTGGTGGAGGCTGAGGAGACTGTGAGAGTGGTGC-3′ and (SEQ IDNO:4) 5′-GACAGATGGTGCAGCCACAGCCCGG TTTATTTCCAACTTTGTCC-3′ for therespective variable regions.

For the human heavy and light chain constant region genes, a humanleukocyte 5′-stretch plus cDNA library was purchased from Clontech (HL5019t). Two PCR reactions were performed on this library using primersspecific for the heavy and light chain constant regions, respectively(Heavy chain: (SEQ ID NO:6)5′-GCACCACTCTCACAGTCTCCTCAGCCTCCACCAAGGGCCCA-3 and (SEQ ID NO:5)5′-ATAAGAATGCGGCCGCTCATTTACCCGGAGACAGGGAG-3′ for full length along with(SEQ ID NO:7) 5′-TGCGGCCGCTCATGGGCACGGTGGGCATGTGT-3′ for FAB generation;Light chain: (SEQ ID NO:9)5′-GGACAAAGTTGGAAATAAACCGGGCTGTGGCTGCACCATCTGTC-3′ and (SEQ ID NO:8)5′-ATAAGAATGCGG CCGCTAACACTCTCCCCTGTTGAAGCT-3′.

A DNA sequence encoding the mouse light chain variable region was fusedin frame to a sequence encoding the human light chain constant region. Afragment encoding the final fusion construct was inserted into P.pastoris expression vector pGAPZ-alpha via ligation through 5′-XhoI and3′-NotI sites in pGAPZ-alpha. DNA sequence encoding the mouse heavyvariable region was fused in frame to sequences encoding each of thethree human heavy chain constant regions. These fusion products werethen inserted using a similar 5′-XhoI and 3′-NotI strategy intopGAPZ-alpha. The proper antibody gene DNA sequences in all vectors wereconfirmed by direct DNA sequencing prior to further work.

Transformation of expression vectors into haploid ade1 ura3, met1 andlys3 host strains of P. pastoris. All methods used for transformation ofhaploid P. pastoris strains and genetic manipulation of the P. pastorissexual cycle were as described in Higgins, D. R., and Cregg, J. M., Eds.1998. Pichia Protocols. Methods in Molecular Biology. Humana Press,Totowa, N.J.

Prior to transformation, each expression vector was linearized withinthe GAP promoter sequences with AvrII to direct the integration of thevectors into the GAP promoter locus of the P. pastoris genome. Samplesof each vector were then individually transformed into electrocompetentcultures of the ade1, ura3, met1 and lys3 strains by electroporation andsuccessful transformants were selected on YPD Zeocin plates by theirresistance to this antibiotic. Resulting colonies were selected,streaked for single colonies on YPD Zeocin plates and then examined forthe presence of the antibody gene insert by a PCR assay on genomic DNAextracted from each strain for the proper antibody gene insert and/or bythe ability of each strain to synthesize an antibody chain by a colonylift/immunoblot method (Wung et. al. Biotechniques 21 808-812 (1996).Haploid ade1, met1 and lys3 strains expressing one of the three heavychain constructs were collected for diploid constructions along withhaploid ura3 strain expressing light chain gene. The haploid expressingheavy chain genes were mated with the appropriate light chain haploidura3 to generate diploid secreting protein.

Mating of haploid strains synthesizing a single antibody chain andselection of diploid derivatives synthesizing tetrameric functionalantibodies. To mate P. pastoris haploid strains, each ade1 (or met1 orlys3) heavy chain producing strain to be crossed was streaked across arich YPD plate and the ura3 light chain producing strain was streakedacross a second YPD plate (˜10 streaks per plate). After one or two daysincubation at 30° C., cells from one plate containing heavy chainstrains and one plate containing ura3 light chain strains weretransferred to a sterile velvet cloth on a replica-plating block in across hatched pattern so that each heavy chain strain contained a patchof cells mixed with each light chain strain. The cross-streaked replicaplated cells were then transferred to a mating plate and incubated at25° C. to stimulate the initiation of mating between strains. After twodays, the cells on the mating plates were transferred again to a sterilevelvet on a replica-plating block and then transferred to minimal mediumplates. These plates were incubated at 30° C. for three days to allowfor the selective growth of colonies of prototrophic diploid strains.Colonies that arose were picked and streaked onto a second minimalmedium plate to single colony isolate and purify each diploid strain.The resulting diploid cell lines were then examined for antibodyproduction.

Putative diploid strains were tested to demonstrate that they werediploid and contained both expression vectors for antibody production.For diploidy, samples of a strain were spread on mating plates tostimulate them to go through meiosis and form spores. Haploid sporeproducts were collected and tested for phenotype. If a significantpercentage of the resulting spore products were single or doubleauxotrophs we concluded that the original strain must have been diploid.Diploid strains were examined for the presence of both antibody genes byextracting genomic DNA from each and utilizing this DNA in PCR reactionsspecific for each gene.

Fusion of haploid strains synthesizing a single antibody chain andselection of diploid derivatives synthesizing tetrameric functionalantibodies. As an alternative to the mating procedure described above,individual cultures of single-chain antibody producing haploid ade1 andura3 strains were spheroplasted and their resulting spheroplasts fusedusing polyethylene glycol/CaCl₂. The fused haploid strains were thenembedded in agar containing 1 M sorbitol and minimal medium to allowdiploid strains to regenerate their cell wall and grow into visiblecolonies. Resulting colonies were picked from the agar, streaked onto aminimal medium plate, and the plates incubated for two days at 30° C. togenerate colonies from single cells of diploid cell lines. The resultingputative diploid cell lines were then examined for diploidy and antibodyproduction as described above.

Purification and analysis of antibodies. A diploid strain for theproduction of full length antibody was derived through the mating ofura3 light chain strain 2252 and lys3 heavy chain strain 2254 using themethods described above. Culture media from shake-flask or fermentercultures of diploid P. pastoris expression strains were collected andexamined for the presence of antibody protein via SDS-PAGE andimmunoblotting using antibodies directed against heavy and light chainsof human IgG, or specifically against the heavy chain of IgG. The datais shown in FIG. 2.

To purify the yeast secreted antibodies, clarified media from antibodyproducing cultures were passed through a protein A column and afterwashing with 20 mM sodium phosphate, pH 7.0, binding buffer, protein Abound protein was eluted using 0.1 M glycine HCl buffer, pH 3.0.Fractions containing the most total protein were examined by Coomasieblue strained SDS-PAGE and immunoblotting for antibody protein.Fractions were also examined via an ELISA assay in which microtiterplates were first coated with F(ab′)2 goat anti-human IgG, Fcγ (JacksonImmuno, Cat No. 109-006-008). Next plates were reacted with selecteddilutions of yeast made antibodies. Finally, plates were reacted withHRP-conjugated goat anti-human F(ab′)2 fragment of IgG F(ab′)2 (JacksonImmuno, Cat No. 109-036-097). Plates were then developed with TMPsubstrate (Sigma Chemical) and reactions were quenched with 0.5 M HCl.Results were quantitated on a BioRad microtiter plate reader at 415 nm.The data is illustrated in FIG. 3.

Assay for antibody activity. The recombinant yeast-derived chimericantibody was evaluated for functional activity throughimmunohistochemical staining of cells containing the target antigen. Thechimeric antibody selectively recognizes the CD3 complex found on Tcells. Jurkat T cells were employed as a source of antigen and cellsurface staining was conducted using procedures described in Anderssonand Sander (Immunol Lett. Jan. 31, 1989; 20(2):115-20) or Andersson et.al. (Eur J Immunol. December 1988; 18(12):2081-4).

Jurkat T cells were immobilized on glass slides, blocked with theappropriate blocking serum and stained with mammalian and yeastgenerated recombinant primary antibody for 1 hour. The immobilizedsamples were then treated with peroxidase blocking agent followed bystaining with a biotinylated Fc selective secondary antibody that isspecific for each form of the antibody (anti-mouse for the mammalian andanti-human for the yeast). Detection was performed using aHRP-Streptavidin system. Digital imaging was performed to collect thedata for each stained sample. Positive signal is detected in samples viaa dark staining of the cells observed in the panels formammalian-derived and yeast-derived OKT-3. This is data is shown in FIG.4.

1. A polyploid Pichia yeast culture which is cultured under conditionswhereby the polyploid yeast culture expresses and secretes into theculture medium at least 10-25 mg/liter to about 1 g/L of at least oneheterologous multichain polypeptide encoded by a heterologous DNA whichis expressed by polyploidal Pichia yeast cells contained in saidpolyploid Pichia yeast culture, wherein the heterologous multichainpolypeptide is an antibody or antibody fragment that specifically bindsto a target antigen.
 2. The polyploid yeast culture of claim 1 whichstably expresses about 25-50 mg/liter of said antibody or antibodyfragment.
 3. The polyploid yeast culture of claim 1 which stablyexpresses about 50-100 mg/liter of said antibody or antibody fragment.4. The polyploid yeast culture of claim 1 which stably expresses about100-250 mg/liter of said antibody or antibody fragment.
 5. The polyploidyeast culture of claim 1 which stably expresses about 1 g/liter of saidantibody or antibody fragment.
 6. The polyploid yeast culture of claim 1which stably expresses about 250-1000 mg/liter of said antibody orantibody fragment.
 7. The polyploid yeast culture of claim 1 which isdiploid.
 8. The polyploid yeast culture of claim 1 which expresses andsecretes more than one antibody or antibody fragment into the culturemedium.
 9. The polyploid yeast culture of claim 1 wherein said antibodyor antibody fragment is selected from the group consisting of an intactantibody, a chimeric antibody, a fully human antibody, a bispecificantibody, a Fab, a Fab2, a camelbody, and a Fab′.
 10. The polyploidalyeast culture of claim 1 which is Pichia pastoris, Pichia angusta orPichia methanolica.
 11. The polyploidal yeast culture of claim 1 whichcomprises at least one mutation which results in enhanced growth orheterologous gene expression or secretion relative to a yeast which doesnot comprise said at least one mutation.
 12. The polyploid Pichia yeastculture of claim 1 wherein the density of Pichia in the culture is atleast 100 g/L.
 13. The polyploid Pichia yeast culture of claim 1 whereinthe density of Pichia in the culture is at least 200 g/L.
 14. Thepolyploid Pichia yeast culture of claim 1 wherein the density of Pichiain the culture is at least 300 g/L.
 15. The polyploid Pichia yeastculture of claim 1 wherein the density of Pichia in the culture is atleast 400 g/L.
 16. The polyploid Pichia yeast culture of claim 1 whereinthe density of Pichia in the culture is at least 500 g/L.
 17. Thepolyploid Pichia yeast culture of claim 12 which expresses and secretesinto the culture medium at least 25-50 mg/liter to about 1 g/L of saidat least one antibody or antibody fragment.
 18. The polyploid Pichiayeast culture of claim 13 which expresses and secretes into the culturemedium at least 25-50 mg/liter to about 1 g/L of said at least oneantibody or antibody fragment.
 19. The polyploid Pichia yeast culture ofclaim 14 which expresses and secretes into the culture medium at least25-50 mg/liter to about 1 g/L of said at least one antibody or antibodyfragment.
 20. The polyploid Pichia yeast culture of claim 15 whichexpresses and secretes into the culture medium at least 25-50 mg/literto about 1 g/L of said at least one antibody or antibody fragment. 21.The polyploid Pichia yeast culture of claim 16 which expresses andsecretes into the culture medium at least 25-50 mg/liter to about 1 g/Lof said at least one a antibody or antibody fragment.
 22. The polyploidculture of any one of claims 12-16 that expresses and secretes into theculture from about 250-1000 mg/L of said at least one antibody orantibody fragment.
 23. The polyploid culture of claim 22 which producesan intact antibody.
 24. The polyploid culture of claim 1 which producesan intact antibody.
 25. The polyploid culture of claim 1 wherein saidantibody or antibody fragment is aglycosylated.
 26. The polyploidculture of claim 22 wherein said antibody or antibody fragment isaglycosylated.
 27. The polyploid culture of claim 23 wherein said intactantibody is aglycosylated.
 28. The polyploid culture of claim 24 whereinsaid intact antibody fragment is aglycosylated.